Production and purification of anti-tubercular and anticancer protein from Staphylococcus hominis under mild stress condition of Mentha piperita L.

Journal Pre-proof Production and purification of anti-tubercular and anticancer protein from Staphylococcus hominis under mild stress condition of Mentha piperita L. Ameer Khusro, Chirom Aarti, Paul Agastian

PII:

S0731-7085(19)33048-1

DOI:

https://doi.org/10.1016/j.jpba.2020.113136

Reference:

PBA 113136

To appear in:

Journal of Pharmaceutical and Biomedical Analysis

Received Date:

16 December 2019

Revised Date:

26 January 2020

Accepted Date:

27 January 2020

Please cite this article as: Ameer K, Chirom A, Paul A, Production and purification of anti-tubercular and anticancer protein from Staphylococcus hominis under mild stress condition of Mentha piperita L, Journal of Pharmaceutical and Biomedical Analysis (2020), doi: https://doi.org/10.1016/j.jpba.2020.113136

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Production and purification of anti-tubercular and anticancer protein from Staphylococcus hominis under mild stress condition of Mentha piperita L.

Ameer Khusro*, Chirom Aarti, Paul Agastian** Research Department of Plant Biology and Biotechnology, Loyola College (Affiliated to University of Madras), Nungambakkam, Chennai – 600034, Tamil Nadu, India

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Corresponding authors E-mail *A. Khusro – [email protected] **P. Agastian- [email protected]

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Mob. +91 9444433117

Highlights

Supplementation of M. piperita L. at log phase of S. hominis strain MANF2

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improved protein production. Strain MANF2 synthesized 51293 Da protein under mild stress of M. piperita L.



MALDI-TOF MS/MS identified the protein as proline dehydrogenase-like protein

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in strain MANF2.

Purified protein revealed in vitro anti-tubercular and anticancer traits in a dose

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

dependent manner.



Protein purified under mild stress can certainly be implied as efficacious antitubercular and anticancer agents in future.

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Abstract The present study was investigated to purify and characterize anti-tubercular and anticancer protein from Staphylococcus hominis strain MANF2 under mild stress condition of Mentha piperita L. Initially, the in vitro anti-tubercular activity of strain MANF2 was determined against Mycobacterium tuberculosis H37Rv using luciferase reporter phage (LRP) assay which showed relative light unit reduction (RLU) of >90%. Further, MTT test revealed promising in vitro anticancer trait of strain MANF2 against lung (A549) and colon (HT-29)

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cancer cell lines. Mild stress of M. piperita L. was provided to strain MANF2 at lag and log phase of its growth and the protein production was optimized statistically using central composite design of response surface methodology. Results showed enhanced protein

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production in the medium containing yeast extract (0.5% w/v) and glycerol (1.5% v/v), being supplemented with M. piperita L. (1.5% v/v) at log phase of strain MANF2. Protein was

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purified using standard purification techniques and showed single homogeneous band on

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SDS-PAGE with nominal molecular mass of 51293 Da, as confirmed by MALDI-TOF MS/MS. The N- amino acid sequencing showed homology with proline dehydrogenase (ProDH), thus, the protein was proposed to be new ProDH-like protein in S. hominis. Further,

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LRP test revealed concentration dependent (10–50 µg/mL) in vitro anti-tubercular properties of purified protein with significant RLU reductions of 36.8±0.3 to 78.5±0.4%. The IC50

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values of purified protein against A549 and HT-29 cancer cells were calculated as 42.2 and

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48.4 µg/mL, respectively. In conclusion, protein purified from strain MANF2 under mild stress of M. piperita L can certainly be implied as efficacious anti-tubercular and anticancer agents in future.

Keywords: Anti-tubercular; Anticancer; M. piperita L.; Protein; S. hominis; Stress

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1. Introduction Tuberculosis (TB) is one of the deadliest tropical diseases of 21st century which infects one third of the world's populace. Mycobacterium tuberculosis (M. tuberculosis) is the key pathogen of TB that invades and replicates inside the host’s macrophage [1]. In spite of the discovery of first-line and second-line anti-tubercular drugs, TB continued to ravage the under-developed and developing countries [1]. Likewise, lung cancer or lung carcinoma is a malignant tumor characterized by uncontrolled cell growth in lung tissues. Worldwide in

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2012, lung cancer occurred in 1.8 million people and resulted in 1.6 million deaths [2]. On the other hand, colon cancer is the third most common type of cancer, making up about 10% of all cases. In 2012, there were 1.4 million new cases and 6,94,000 deaths from the disease

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[2]. With the emerging dilemma of multi-drug resistant tuberculosis (MDR-TB) and extensively-drug resistant tuberculosis (XDR-TB) as well as serious side effects of existing

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cancer treatments, the exigency for developing new anti-tubercular and anticancer agents

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through novel approaches is an obligation now for worldwide researchers. Antimicrobial proteins (AMPs) are gene-encoded proteins produced by genera from specific domains, showing direct growth inhibitory activities, and playing significant roles in

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host defense [1]. These AMPs can contribute to mycobactericidal innate immunity through direct as well as indirect (immune modulation) action [1]. In addition, protein/peptide from

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distinct sources has shown interesting potential in cancer therapy, most of them are currently

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undergoing phase III clinical trials in patients [3]. However, studies revealing the antitubercular and anticancer attributes of bacteria associated proteins are limited. Coagulase-negative staphylococci (CNS) are generally non-pathogenic commensals of

humans and have received less attention among scientific communities [4]. Coagulasenegative staphylococci, autochthonous to fermented foods grow as non-infectious bacteria and contribute to the fermentation process [5]. Most importantly, CNS are known to produce

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distinct classes of AMPs as bactericidal agents [6]. Similarly, proteins from varied groups of bacteria have revealed selective toxicity towards disparate cancer cells viz. breast cancer, lung cancer, colon cancer, and bone cancer. Some of them, including anticancer antibiotics (actinomycin D, bleomycin, doxorubicin, and mitomycin C) and diphtheria toxin are already used in the cancer therapy, while other substances are in clinical trials [7]. Bacteria up-regulate and down-regulate many transcripts under stress conditions which lead to affect protein promoter [8]. Antimicrobial agents or antimicrobials are

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pronounced lethal substances exhibiting broad spectrum inhibition of bacterial growth by modulating transcription mechanism in cells. Bacterial metabolism is induced due to the sublethal concentration exposure of certain antimicrobials [9]. Among ideal antimicrobials,

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medicinal plants at low concentrations provide stress to bacteria, thereby causing variations in the physiology and metabolism of the cell [9]. In folk medicine, Mentha piperita L.

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(Family – Lamiaceae) or peppermint has been used as antiemetic, antiparasitic, anti-

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inflammatory, antibacterial, and antispasmodic agents. Phytochemicals such as flavonoids, phenols, and terpenoids present in the leaves of M. piperita L. are mainly responsible for its

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pronounced antibacterial properties [10].

In spite of the exposure of diverse unfavourable conditions such as high temperature,

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salt concentration, acidity, alkalinity, and oxidative stress to bacteria, there are very few studies assessing the potentiality of medicinal plants as signalling agents to trigger biological

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characteristics in bacteria, probably unavailable in fermented foods associated CNS. Considering this, a significant attempt was undertaken in this investigation to purify and characterize bioactive protein from Staphylococcus hominis, synthesized under mild stress of M. piperita L., and to evaluate its in vitro anti-tubercular as well as anticancer properties. Further attempt was also undertaken in this study to understand the hypothetical pathway towards the synthesis of protein under mild stress of M. piperita L. 4

2. Materials and methods 2.1.

Bacterium of interest and growth parameters

Staphylococcus hominis strain MANF2, previously isolated from Koozh (a traditional fermented food of Southern India) was used in this study [11]. Unless otherwise stated, strain MANF2 was grown at 30°C for 48 h in 250 mL Erlenmeyer flasks constituting 50 mL of de Man Rogose Sharpe (MRS) broth (g/L – proteose peptone 10.0, beef extract 10.0, yeast extract 5.0, dextrose 20.0, polysorbate 80 1.0, ammonium citrate 2.0, sodium acetate 5.0,

for further experimental studies. 2.2. In vitro anti-tubercular activity of strain MANF2–

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2.2.1. Cell free neutralized supernatant (CFNS) preparation

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magnesium sulphate 0.1, manganese sulphate 0.05, and dipotassium phosphate 2.0) medium

Strain MANF2 was inoculated into sterilized MRS broth and incubated at 30°C for 48 h.

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Bacteria were centrifuged at 8000 g for 10 min, and the culture supernatant was subjected to

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membrane filtration (0.22 μm). The sterilized cell free supernatant was neutralized (pH 7.0) using 1N sodium hydroxide (NaOH) in order to exclude the anti-tubercular effect of organic acids in the medium. Thus, the CFNS obtained was treated individually with catalase (Sigma,

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India; 1 mg/mL) and incubated at 37°C for 2 h in order to eliminate the inhibitory effect of hydrogen peroxide (H2O2). After catalase treatment, the CFNS obtained was then lyophilized

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and screened for anti-tubercular activity against Mycobacterium tuberculosis H37Rv.

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2.2.2. Preparation of M. tuberculosis suspension The bacterial suspensions were prepared by inoculating the log phase culture of reference

laboratory strain M. tuberculosis H37Rv from Lowenstein-Jensen slope into Middlebrook G7H9 broth (HiMedia Laboratories, India). 2.2.3. Luciferase Reporter Phage (LRP) assay

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Anti-tubercular activity of CFNS of strain MANF2 was determined by adopting LRP assay as described by Sivakumar et al. [12] with slight modifications. Approximately 350 μL of G7H9 broth (HiMedia Laboratories, India) supplemented with 10% albumin dextrose complex and 0.5% glycerol were taken in cryovials, and CFNS (1 mg/mL) was added into it. Hundred microliters of M. tuberculosis suspension was inoculated into all the vials. Sterile double distilled water was used as solvent control. Rifampicin (30 μg/mL) was used as standard drug control. All the vials were incubated at 370C for 72 h. After required period of

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incubation, 50 μL of high titre mycobacteriophage phAETRC202 and 40 μL of 0.1M calcium chloride solution were added into the test as well as control vials. All the vials were incubated at 370C for further 4 h. After incubation, 100 μL from each vial were transferred to

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luminometer cuvette. About 100 μL of D-luciferin (0.3 mM in 0.05M sodium citrate buffer, pH 4.5) was added into it and relative light unit (RLU) was measured in luminometer (Model

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– Monolight 2010). All the readings were recorded in triplicate, and the mean values were

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calculated. Percentage (%) RLU reduction was calculated as followsRLU reduction (%) = RLUControl - RLUTest / RLUControl × 100 In vitro anticancer activity of strain MANF2–

2.3.1. Cell culture

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2.3.

(Equation 1)

Human lung cancer cells (A549) and human colon cancer cells (HT-29) were purchased

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from National Centre for Cell Lines, Pune, India. Cells were maintained in Dulbecco's

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Modified Eagle Medium supplemented with 2 mM L-glutamine and balanced saline adjusted to contain 1.5 g/L sodium carbonate, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine, 1.5 g/L glucose, 10 mM hydroxyethyl piperazineethanesulfonic acid, and 10% fetal bovine serum. Penicillin and streptomycin (100 IU/100 µg) were adjusted to 1 mL/L. Cells were maintained at 37⁰C with 5% carbon dioxide (CO2) in a humidified CO2 incubator.

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2.3.2. In vitro cytotoxicity Anticancer activities of CFNS of strain MANF2 against A549 and HT-29 cells were evaluated using 3-(4, 5-dimetylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay [13]. Cancer cells were grown (1×104 cells/well) in a 96-well plate for 48 h into 75% confluence. The medium was replaced with fresh medium containing protein sample, and cells were further incubated for 48 h. The culture medium was removed, MTT solution (100 µL) was added to each well, and incubated at 37⁰C for 4 h. After removal of the supernatant,

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50 µL of dimethyl sulphoxide was added to each of the wells and incubated for 10 min to solubilise the formazan crystals. The absorbance was measured at 620 nm using an ELISA multi-well plate reader (Thermo Multiskan EX, USA). Doxorubicin (Sigma, India) was used

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as positive control. The absorbance value was used to calculate % viability using the

(Equation 2)

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2.4. Antibacterial agent of interest

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following formula.

M. piperita L. was purchased freshly from the local market of Nungambakkam, Chennai,

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India and stored at 4⁰C. Fresh leaves of M. piperita L. were surface sterilized using 95% (v/v) ethanol, and washed with autoclaved distilled water too. Leaves were homogenized using

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grinder, filtered through six layers of muslin cloth, and then centrifuged at 6000 g for 15 min. The supernatant was collected, filter sterilized using 0.2 µm syringe filter, and considered as

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100% [14].

2.5. Antibacterial activity of M. piperita L. The antibacterial activity of M. piperita L. was determined against strain MANF2 using

the methodology of Khusro and Sankari [9]. Strain MANF2 was inoculated into sterilized MRS broth aseptically and incubated at 30⁰C for 24 h. After required period of incubation, the culture was swabbed into freshly prepared MRS agar medium using sterile cotton swab. 7

On the other hand, commercially available sterile discs (6 mm diameter) were soaked with 25 µL of M. piperita L. (100%) and allowed to dry for 30 min. Discs were placed aseptically over previously prepared MRS agar plate (seeded with the test bacterium) using ethanol dipped and flamed forceps. Plate was incubated at 30⁰C and observed for the zone of inhibition after 48 h. The diameter of zone of inhibition was measured in millimetre (mm) and result was recorded 2.6. Minimum inhibitory concentration (MIC) and sub-MIC determination

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The MIC and sub-MIC values of M. piperita L. were determined against strain MANF2 using microdilution assay as per the methodology of Khusro and Sankari [9] with slight modifications. Fifty microlitres of overnight grown bacterial inoculums was added into each

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well of the microtitre plate containing 50 μL of M. piperita L. as per the dilutions (100-1% concentrations) prepared (data not shown). Dilutions of M. piperita L. were made using MRS

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broth. The well containing only bacterial inoculums was considered control. The microtitre

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plate was incubated at 30°C for 48 h. After required incubation period, the viability of bacteria was observed by adding 40 µL of MTT solution as an indicator into each well of the

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microtitre plate. The microtiter plate was further incubated at 30°C for 30 min and observed for colour change. The colour change of the suspension from yellow to dark purple indicates the bacterial growth, while yellow colour in remaining wells indicates lack of bacterial

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growth. The lowest concentration of M. piperita L. inhibiting the growth of strain MANF2

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was considered as MIC value. Half of the value of the MIC was calculated as sub-MIC of M. piperita L.

2.7. Growth curve analysis of strain MANF2 Growth curve was analyzed to determine the lag and log phase of strain MANF2. One millilitre of culture was inoculated aseptically into freshly prepared MRS broth and incubated

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at 30⁰C under shaking condition. The absorbance was read at 600 nm using UV-Vis spectrophotometer every 1 h interval [15]. 2.8. M. piperita L. stress M. piperita L. at sub-MIC concentration was used to provide stress during lag and log phase of strain MANF2, as determined through growth curve. The MRS broth medium was autoclaved and overnight grown culture of strain MANF2 was added into it aseptically. M. piperita L. at sub-MIC value was added into the culture broth during lag and log phase of

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bacteria. Flasks were incubated at 30⁰C under shaking condition and protein production was estimated after 48 h according to Bradford assay [16] using bovine serum albumin as standard.

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2.9. Statistical optimization of protein production under M. piperita L. stress

Central composite design (CCD) of response surface methodology (RSM) was

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employed to optimize three parameters (yeast extract, glycerol, and M. piperita L.) in order to

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enhance extracellular protein production from strain MANF2 under M. piperita L. stress. Parameters such as yeast extract and glycerol were selected for statistical optimization based

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on their significant impact on protein production from strain MANF2 obtained via one factor at a time method (data not shown). Each variable in the design matrix was tested at five different levels (-α, -1, 0, +1, +α). The design involved 6 central points with an alpha (α)

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value being ±2. The total number of experimental combinations is 2k + 2k + n, where ‘k’ is

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the number of variables and ‘n’ is the number of repetition of experiments at the central point. The experimental design consisted of 20 runs of three variables for enhancing the protein production under mild stress of M. piperita L. All variables were set at a central coded value of zero. The efficacy of selected variables to the response was calculated by a second-order polynomial equation as given belowY (mg/mL) = β0 + β1A + β2B + β3C + β11A² + β22B² + β33C² + β12AB + β13AC + β23BC

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(Equation 3) where, Y is dependent variable (protein production) β0 is intercept β1, β2, β3 are linear coefficients β11, β22, β33 are squared coefficients β12, β13, β23 are interaction coefficients A, B, C, A², B², C², AB, AC, and BC are variables’ levels.

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The accuracy of polynomial model was inferred by R2. The parameters affecting significantly the production of protein from strain MANF2 was analyzed by Analysis of variance (ANOVA).

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2.10. Purification of protein

In this study, strain MANF2 exhibited maximum production of protein due to the

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supplementation of mild concentration of M. piperita L. at log phase of bacterial growth.

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Hence the protein was purified from strain MANF2 after inoculation of M. piperita L. (1.5% v/v) during log phase of bacterial growth only. 2.10.1. Medium preparation

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One litre of MRS broth medium was prepared using statistically optimized parameters and autoclaved. The overnight grown culture of strain MANF2 was inoculated into cooled

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MRS broth medium aseptically and incubated at 30⁰C under shaking conditions. M. piperita

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L. (1.5% v/v) was added into the bacterial culture during its log phase of growth and incubated up to 48 h. 2.10.2. Ammonium sulphate precipitation The bacterial culture was centrifuged at 8000 g for 15 min at 4⁰C. The supernatant was collected, neutralized, and precipitated with powdered ammonium sulphate (60% saturation) at 4⁰C. The precipitated protein samples were collected after centrifugation at 10000 g for 15 10

min at 4⁰C. The resulting pellets were re-suspended in sodium phosphate buffer (0.1M, pH 7.0) for the determination of total protein content using Bradford assay [16]. 2.10.3. Dialysis Dialysis membrane (1.0 kDa cut-off) was pre-treated using freshly prepared 10 mM ethylenediaminetetraacetic acid (EDTA), 2% (w/v) sodium bicarbonate (NaHCO3), and distilled water (65⁰C as well as room temperature). The dialysis membrane was dipped into the solutions in the sequential order of distilled water (65⁰C) for 5 min, 10 mM EDTA for 5

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min, 2% (w/v) NaHCO3 for 5 min, and distilled water (room temperature) for 5–10 min. The sample containing membrane was dipped into approximately 1 L of sodium phosphate buffer (0.1M, pH 7.0) and kept under slow stirring at cold condition for 2–3 h. The buffer was

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changed every 2–3 h and continued the process for 4 cycles under cold condition. Total protein content of dialysed sample was estimated according to Bradford assay [16] and used

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for further process.

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2.10.4. DEAE–Cellulose column chromatography

DEAE-Cellulose (1.5 g) was soaked in 0.1M (pH 7.0) phosphate buffer. The column was

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washed using distilled water and then with the buffer. The column was packed with presoaked DEAE cellulose and excess buffer was eluted. The dialyzed sample was loaded into

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the column. After 30 min, sample was eluted using the same buffer at the flow rate of 0.5 mL/min. The gradient elution was carried out using 0.2, 0.3, 0.4, 0.5M sodium chloride in the

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same buffer. Fractions were collected and tested for in vitro anti-tubercular and anticancer activities according to the methodology as discussed earlier in this study. Fraction showing maximum activity was electrophoresed for determining its molecular mass. 2.10.5. Molecular mass determination The purity and molecular mass of the protein sample were determined using Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) with 4% stacking gel and 11

12% separating gel. Ammonium sulphate precipitated, dialyzed, and purified protein samples were mixed with sample solubilising buffer (1:1) and the mixture was heated at 950C in water bath for 10 min. Protein sample along with standard protein marker was then loaded into the SDS-PAGE. The gel was electrophoresed at 50–100V for 3–4 h in order to allow the samples run completely into the gel. After electrophoresis, the gel was placed in coomassie brilliant blue staining solution for 1 h and washed thoroughly with distilled water. The stained gel was then placed in destaining solution (methanol: acetic acid: distilled water – 5:1:4). Destaining

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solution was changed every 30 min and the destaining was continued till the bands are clearly observed. Protein band was observed and its molecular mass was calculated based on the relative mobility of the standard marker (14.3–66 kDa).

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2.10.6. MALDI-TOF MS/MS and N- terminal amino acid sequencing

The single band of purified protein was excised from the SDS-PAGE gel and kept in 50%

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(v/v) methanol. Matrix assisted laser desorption ionization-time of flight mass spectrometry

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(MALDI-TOF MS/MS) was used to determine the molecular mass of the purified protein. The protein was digested using trypsin at 37⁰C for 12 h. An equal volume of cyano-4-

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hydrocinnamic acid solution (10 mg/mL CHCA in 70% acetonitrile, 0.03% trifluoro acetic acid) was added to the trypsinized protein. MALDI-TOF MS/MS spectrum depicts the fragmentation of protein into various peptides. Peptide mass fingerprinting (PMF) was

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carried out to examine the sequence of the peptide fragments of protein. The N- amino acid

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sequence was compared with the sequence available in BLAST, NCBI algorithm (http://www.ncbi.nlm.nih.gov/)

with

the

MASCOT

search

program

(http://matrixscience.com). 2.11. Fourier transform infra-red (FT-IR) spectroscopy Three milligrams of lyophilized protein sample were mixed with 300 mg of potassium bromide (FT-IR grade) and pressed into a pellet in a hydraulic press by applying 500 kg/m3

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pressure. The pellet was put into the sample holder and FT-IR spectra were recorded ranging 4000–400 cm−1 using FT-IR spectroscopy [Model No.- IRAffinity- 1(SHIMADZU)]. 2.12. In vitro anti-tubercular activities of purified protein– 2.12.1. Concentration dependent anti-tubercular activities M. tuberculosis H37Rv was incubated in the presence of various concentrations (10– 50 µg/mL) of purified protein and the anti-tubercular activities were estimated according to the LRP assay as mentioned earlier in this study using rifampicin as positive control.

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2.12.2. Effect of pH on anti-tubercular activities of purified protein

pH of the purified protein was adjusted from 3.0 to 8.0 using 1N hydrochloric acid and 1N NaOH. Samples were incubated at 37⁰C for 3 h and residual anti-tubercular activities

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were calculated against the control (pH 6.5) according to the methodology as mentioned

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earlier in this study.

2.12.3. Effect of temperature on anti-tubercular activity of purified protein

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To investigate the thermal effect on residual anti-tubercular activities, purified protein at optimum pH level was incubated at 40⁰C for 2 h, 60⁰C for 1 h, 80⁰C 30 min, 100⁰C for 20

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min, and 121⁰C for 15 min. The protein sample without heat treatment was considered as control. The residual anti-tubercular activities were calculated according to the methodology

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as mentioned earlier in this investigation. 2.12.4. Effect of enzymes on anti-tubercular activities of purified protein

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Enzymes (1 mg/mL; pepsin – pH 3.0, trypsin – pH 7.5, protease K – pH 7.5, and α-

amylase – pH 7.5) were mixed with purified protein at optimal pH and temperature. The mixture was incubated for 1 h and then subsequently heated at 100⁰C for 5 min in order to inactivate the enzymes. Residual anti-tubercular activities were calculated according to the methodology as mentioned earlier in this study. The purified protein without the addition of enzymes was used as control. 13

2.13. In vitro anticancer activities of purified protein Anticancer activities of various concentrations (10–50 µg/mL) of purified protein were determined against A549 and HT-29 cancer cells using MTT assay according to the methodology as mentioned earlier in this investigation. The minimum concentration of protein required for 50% inhibition of cell viability (IC50) was calculated using linear regression curve statistics. 2.14. Statistical analyses and optimization software used

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Experiments were demonstrated in triplicate and values were expressed in terms of mean±SD. The statistical optimization process was interpreted using Design Expert Version 11.0.0 (Stat-Ease Inc., Minneapolis, Minnesota, USA) software. Data were validated using

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ANOVA and values P≤0.05 were considered statistically significant. 3. Results

Anti-tubercular and anticancer activities of strain MANF2

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3.1.

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The CFNS of strain MANF2 exhibited pronounced anti-tubercular activity against M. tuberculosis H37Rv with RLU reduction of >90% (Figure not shown). On the other hand, the

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CFNS of strain MANF2 showed comparatively higher anticancer activity against A549 cancer cells as compared to HT-29 cells. The viability of A549 and HT-29 cancer cell lines was estimated as 76.5±0.4 and 84.2±0.3%, respectively (Figure not shown). Antibacterial activity of M. piperita L. and sub-MIC determination

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3.2.

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M. piperita L. exhibited promising antibacterial activity against strain MANF2 with 20.3±0.03 mm of zone of inhibition (Figure not shown). The MIC value of M. piperita L. against strain MANF2 is shown in Fig. 1 which was determined as 25%. Thus, the sub-MIC value of M. piperita L. against strain MANF2 was estimated as 12.5% (Table 1). 3.3.

Growth curve analysis of strain MANF2

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Fig. 2 shows the growth curve of strain MANF2. The lag and log phase of the bacterium was achieved at 2 and 9 h. M. piperita L. stress was provided to the bacterium at lag and log phase, and protein production was estimated after statistical optimization. 3.4.

Statistical optimization of protein production under M. piperita L. stress

Shake flask fermentation was carried out to provide M. piperita L. stress to strain MANF2 during lag and log phase of its growth. Protein production from strain MANF2 under mild stress of M. piperita L. was optimized using CCD of RSM. Table 2 shows

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selected variables with their respective ranges employed in CCD tool. Table 3A represents the CCD matrix of selected variables in coded units along with observed as well as predicted values of protein production under mild stress of M. piperita L., provided at lag phase of

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strain MANF2

The response (Y) optimized through CCD was estimated by the following model

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equation:

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Y (mg/mL) = 3.04 + 1.02A – 0.5009B + 1.08C – 0.6307AB + 0.5211AC + 0.1693BC + 0.2324A2 – 0.0935B2 + 0.3738C2

(Equation 4)

A total of 20 experiments were conducted at diverse combinations of selected

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variables and the maximum protein production of 6.4 mg/mL was achieved from Run No. 14, consisting of medium with yeast extract (0.5% w/v), glycerol (1.5% v/v), and M. piperita

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(1.5% v/v). The experimental protein production from strain MANF2 was observed close to

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the predicted value (6.2 mg/mL). Table 3B represents ANOVA for protein production quadric model at lag phase of

bacterial growth. The model F-value of 48.47 implies the model is significant. There is only 0.01% chance that a large “Model F-value” could occur due to noise. Values of “Prob > F” < 0.05 indicate model terms are significant. In this study, A, B, C, AB, AC, A2, and C2 are significant model terms. The “Lack of Fit F-value” of 1.02 implies the Lack of Fit is non-

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significant relative to the pure error. Non-significant lack of fit is good for the model to fit. The R2 (0.9776) indicates appropriate correlation between experimental and predicted response values, and thus reveals the accuracy of the model. In this study, a low C.V. (8.62%) represents the reliability of the experiments. The “Predicted R2” of 0.8834 is in reasonable agreement with the “Adj R2” of 0.9574. “Adeq Precision” ratio of 22.52 indicates an adequate signal, and thereby suggests the navigation of design space. Table 4A represents the CCD matrix of selected variables in coded units along with

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observed as well as predicted values of protein production under mild stress of M. piperita L., provided at log phase of strain MANF2.

The response (Y) optimized through CCD was estimated by the following model

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equation:

0.1172A2 – 0.1317B2 + 0.2586C2

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Y (mg/mL) = 6.03 + 1.22A – 0.6139B + 1.33C – 0.6974AB + 0.4253AC + 0.1776BC + (Equation 5)

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A total of 20 experiments were conducted at diverse combinations of selected variables and the maximum protein production of 9.4 mg/mL was achieved from Run No. 14, consisting of medium with yeast extract (0.5% w/v), glycerol (1.5% v/v) and M. piperita L.

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(1.5% v/v). The experimental protein production from strain MANF2 was estimated close to the predicted value (9.3 mg/mL).

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Table 4B represents ANOVA for protein production quadric model at log phase of

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bacterial growth. The model F-value of 66.73 implies the model is significant. There is only 0.01% chance that a large “Model F-value” could occur due to noise. Values of “Prob > F” < 0.05 indicate model terms are significant. In this study, A, B, C, AB, AC, and C2 are significant model terms. The “Lack of Fit F-value” of 0.981 implies the Lack of Fit is nonsignificant relative to the pure error. Non-significant lack of fit is good for the model to fit. The R2 (0.9836) indicates appropriate correlation between experimental and predicted

16

response values, and thus reveals the accuracy of the model. In this study, a low C.V. (4.66%) represents the reliability of the experiments. The “Predicted R2” of 0.9208 is in reasonable agreement with the “Adj R2” of 0.9689. “Adeq Precision” ratio of 26.29 indicates an adequate signal, and thereby suggests the navigation of design space. The optimized conditions obtained using CCD were used for the authentication experiment. The observed protein production of strain MANF2 was estimated very much close to the predicted value, thereby indicating the authentication of model towards the

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improvement of protein production. In brief, medium containing yeast extract (0.5% w/v) and glycerol (1.5% v/v), being supplemented with M. piperita L. (1.5% v/v) at log phase of strain MANF2 revealed enhanced protein production. Purification of protein

-p

3.5.

The anti-tubercular protein was purified using standard protein purification techniques

re

including ammonium sulphate precipitation, dialysis, and DEAE–Cellulose column

lP

chromatography. The summary of purification steps is shown in Table 5. Proteins precipitated at 60% saturation showed relatively higher total protein content. The protein precipitated with 60% saturation was used for dialysis which revealed total protein content of

further

purified

na

340.3 mg with 79.1% recovery and purification fold of 29.1. The dialysed product was using

DEAE–Cellulose

column

chromatography.

After

column

ur

chromatography, the yield and purification fold of protein were estimated to be 72.3% and

Jo

1372.1, respectively. A total of 16 fractions were collected from the elution process and tested for anti-tubercular and anticancer activities. Among all the fractions tested, fraction 6 showed maximum anti-tubercular and anticancer activities (data not shown). 3.6.

Molecular mass determination

17

The purity and molecular mass of the purified protein were observed using SDS-PAGE which showed a single homogeneous band corresponding to the apparent molecular mass of 52 kDa based on relative mobility (Fig. 3). 3.7.

MALDI-TOF MS/MS spectrum and N- terminal amino acid sequencing Fig. 4 shows MALDI-TOF MS/MS spectrum of purified protein. The nominal mass

of the purified protein was found to be 51293 Da as obtained from PMF analysis. Each peak in the spectrum represents peptides present in the tryptic digest of purified protein. The size

ro of

of the peptide ion varies from m/z 1689.63 to 3555.06. The most abundant peptide ion is m/z 2110.87. Identification of protein using database which contains theoretical tryptic digests of all known proteins revealed the sequence homology (100%) with 1–1321 amino acids of

-p

proline dehydrogenase (ProDH). The amino acid sequence of the most abundant peptide (m/z = 2110.87) in the purified protein was identified as ‘Leu-Val-Ser-Thr-His-Asn-Glu-Ala-Gly-

FT-IR spectroscopy

lP

3.8.

re

Leu-Thr-Ser-Ser-Leu-Asn-Arg-Leu-Ile-Gly-Lys’ (LVSTHNEAGLTSSLNRLIGK).

The FT-IR spectrum of the purified protein is shown in Fig. 5. The high absorption bands at 3422.2, 2966.9, 2344.72, 1641.32, 1451.9, and 1105.75 cm-1 indicated the presence of

3.9.

na

strong N-H stretch, C-H bond of amide I, C-N bond, C=O stretch, and C-H bending. In vitro anti-tubercular activities of purified protein

ur

The anti-tubercular activities of purified protein were increased in a concentration

Jo

dependent manner (10–50 µg/mL) ranging from RLU reduction of 36.8±0.3 to 78.5±0.4% with respect to the standard (75.5±0.5 to 99.9±0.5%) (Fig. 6). 3.10.

Characterization of purified protein

The impact of pH, temperature, and enzymes on the residual anti-tubercular activities of purified protein is shown in Table 6. The anti-tubercular activities of protein varied significantly (P<0.05) at various pHs. The protein retained its activity at pH 6.0 and 7.0 with

18

residual activities of 93.5±0.5 and 96.6±0.4%. The residual anti-tubercular activities of protein were reduced at acidic and alkaline pHs. The heat treatment on purified protein revealed significant (P<0.05) variations in residual anti-tubercular activities. The protein retained maximum residual activity of 97.2±0.4% at 40⁰C for 2 h. The residual antitubercular activities were drastically reduced (10.8±0.4%) at high temperature (121⁰C for 15 min). The anti-tubercular activities of purified protein were lost completely after treatment

amylase, showing residual activity of 98.2±0.5%. 3.11.

In vitro anticancer activities of purified protein

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with pepsin, trypsin, and protease K. On the other hand, the protein was found resistant to α-

The anticancer activities of protein against A549 cancer cell line is shown in Fig. 7.

-p

After treatment with purified protein at various doses, cell shrinkage followed by rounding of cells and reduction in cell counts occurred. The viabilities of A549 cells were estimated

re

(92.6±0.3–41.3±0.4%) to be reduced significantly (P<0.05) with increase in the protein

lP

concentrations. A similar morphological alteration was observed in HT-29 cancer cells after treatment with purified protein (Fig. 8). The viabilities of HT-29 cell lines was also estimated

na

(94.6±0.3–48.5±0.4%) to be reduced significantly (P<0.05) with increase in the protein concentrations. The purified protein revealed comparatively higher anticancer activity against

ur

A549 cells with respect to HT-29 cells. The IC50 values of purified protein against A549 and HT-29 cancer cells were calculated as 42.2 and 48.4 µg/mL, respectively. In contrary to this,

Jo

doxorubicin exhibited pronounced anticancer activities with IC50 values of 7.4 and 9.6 µg/mL against A549 and HT-29 cancer cells, respectively. 4. Discussion

The existing scenario of anti-tubercular therapies as well as uncontrollable emergence of MDR-TB and XRD-TB has drawn immense attention to achieve competent and durable treatment of TB. Despite limited studies, bacterial proteins have been recognized as one of

19

the most important biological macromolecules exhibiting promising anti-tubercular traits in the past. Previously, bacteriocins viz. nisin and lacticin had shown significant growth inhibitory activities against M. tuberculosis [17]. In the present study, CFNS of strain MANF2 showed promising in vitro anti-tubercular activity against M. tuberculosis H37Rv, thereby suggesting the potency of fermented food associated CNS as ideal anti-tubercular agents. Over the past few years, bacterial proteins and peptides have also been investigated as

ro of

promising anticancer agents. Karpinski and Adamczak [18] reviewed exemplary tumoricidal role of bacterial supernatants and proteins against distinct types of cancer cells. Although diversiform groups of bacteria are known to secrete a wide array of tumoricidal agents, little

-p

is known about the anticancer attributes of fermented food associated CNS, probably undetermined yet. In this context, the CFNS of strain MANF2 showed pronounced in vitro

re

anticancer activities against lung cancer and colon cancer cell lines. The findings

lP

demonstrated the anticancer role of strain MANF2 against A549 and HT-29 cell lines, and emphasized us to investigate further the CNS strains as ideal anticancer agents. Bacteria are exposed to diversified kinds of stresses in the environment which impair

na

their metabolism. The potentiality to adapt rapidly the changing environmental conditions is one of the most important hallmarks of bacterial survival ability. Mild or drastic variations in

ur

the environment can cause alteration in the transcriptome of bacterial cells [9]. As a matter of

Jo

fact, stress can elicit dramatic variations in the expression pattern of stress-related genes encoding proteins which improve adaptation toward the changing environment. The sensing of any kind of stress sends alarming signals to bacteria, and thus, the mechanisms within them start working overtime to neutralize or pump out the undesired environment as well as to repair damaged constituents of the cell. This situation triggers transcriptional variations in the bacteria and produces or secretes new proteins [14].

20

In this study, antimicrobial agent (M. piperita L.) at sub-MIC concentration was used to provide stress to strain MANF2 during the lag and log phase of bacterial growth. Mild concentration of M. piperita L. supplemented at log phase of strain MANF2 showed significant (P<0.05) enhancement in protein production using statistical optimization tool. The finding suggested that the protein production from bacteria can be enhanced by supplementing the particular doses of antimicrobials, particularly M. piperita L. at specific growth period of bacteria. It was supported by the previous studies of Ismail et al. [14] and

ro of

Khusro and Sankari [9] who evaluated paramount role of Allium sativum in enhancing and synthesizing novel protein in Lactobacillus sp. and Bacillus sp., respectively using nonstatistical tool. Our findings revealed first report on the promising role of M. piperita L. in

-p

enhancement of protein production from fermented food associated coagulase-negative Staphylococcus sp.

re

Extracellular stimulations cause alterations in the metabolic activities of bacteria [8, 9]. In

lP

the present investigation, strain MANF2 showed rapid variations in their cellular responses after exposure of M. piperita L. at mild concentrations. The mild stress of antimicrobial agents might induce the production of metabolism associated distinct proteins. It should be

na

noteworthy that bacteria obtain energy for growth through metabolic processes [14]. A stimulus can over-express a variety of genes which produce metabolism related new proteins

ur

inside the bacterial cells [9]. An immediate defensive response is required for the survival of

Jo

bacteria in the presence of external stimuli. In this regard, translational regulation of preexisting mRNAs provides a fast and efficient strategy for controlling the expression of various genes [14]. Notably, the stress-induced attenuation of global translation is often accompanied by a switch to the selective translation of proteins that are essential for the survival of bacteria under stress [19]. In the current study, protein involved in the translation

21

process were differentially expressed by strain MANF2 following exposure to M. piperita L. stress at log phase of bacterial growth. Strain MANF2 under M. piperita L. stress produced anti-tubercular protein of molecular mass 51293 Da. Based on amino acid sequencing, it showed homology with ProDH, thereby categorizing it as new class of ProDH- like protein in S. hominis. Findings established the fact that because of stress, translation is regulated to mitigate energy consumption and to synthesize proteins selectively for the proper establishment of tolerance. Reports on the

ro of

purification of ProDH from bacterial sources are sparse. Previous studies have revealed the purification of ProDH of varied molecular masses from Escherichia coli [20], Pseudomonas entomophila [21], and Pseudomonas putida [22]. Interestingly, our findings represented the

-p

first report on the purification of ProDH from S. hominis under mild stress condition of antimicrobial agent.

re

Oxidation of amino acids is a central part of energy metabolism. Proline is a

lP

proteinogenic secondary amino acid, which plays essential roles not only in primary metabolism but also in redox homeostasis, osmotic adjustment, protection against stress, and signalling in all organisms [23]. The chemical reactions of the proline metabolic pathway are

na

shown in Fig. 9. Proline is synthesized from glutamate starting with the enzymes glutamate kinase (GK) and γ-glutamyl phosphate reductase (GPR), which in plants and animals is fused

ur

together in the bifunctional enzyme ∆1-pyrroline-5-carboxylate synthetase (P5CS). The

Jo

intermediate γ-glutamate-semialdehyde (GSA) spontaneously cyclises to ∆1-pyrroline-5carboxylate (P5C), which is then reduced to proline by P5C reductase (P5CR). Alternatively, proline can be formed from ornithine, which is converted into P5C/GSA via ornithine-δaminotransferase (OAT) [24]. Proline acts as energy supplier and may stabilize DNA, protein, and membrane structure and function of plants. M. piperita L. contains promising amount of proline, as investigated

22

by Askary et al. [25] and Samandari-Gikloo et al. [26]. Despite the presence of proline originally into the plant, proline accumulation in plants also occurs due to various stresses, including salt, drought, ultra-violet radiation, and heavy metal ions [24]. Besides, proline accumulation is observed in response to environmental stress in bacteria too [27]. In this study, M. piperita L. was used as antibacterial agent to provide mild stress to strain MANF2, which clearly indicated the growth of bacteria in the presence of proline. The oxidative pathway for proline degradation consists of two enzymatic steps and an intervening non-

ro of

enzymatic equilibrium. ProDH and Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH) catalyze L-proline into glutamate. The first enzymatic step transforms proline to P5C, which is non-enzymatically hydrolyzed to glutamic semialdehyde. The semialdehyde is oxidized in

-p

the second enzymatic step to glutamate (Fig. 10) [22].

ProDH catalyzes the oxidation of L-proline to P5C leading to the release of electrons,

re

which can be transferred to either electron transfer systems or to molecular oxygen. ProDH is

lP

essential not only for proline catabolism but also provides energy [28]. In most bacteria, ProDH and P5CDH activities are combined in a single bifunctional enzyme known as Proline utilization A (PutA). Studies of the bacterial enzymes have shown that ProDH is an FAD-

na

dependent enzyme [28].

The hypothetical pathway for the production of ProDH from strain MANF2 under mild

ur

stress of M. piperita L. is proposed in Fig. 11. The supplementation of mild concentration of

Jo

M. piperita L. exposed the bacterium to proline present in this plant, as synthesized through proline metabolism pathway. The ProDH encoding gene might have been ‘switched on’ through the signalling mechanism due to the presence of external stimulus. ProDH was secreted into the medium to catalyze the proline oxidation mechanisms; thereby the production of extracellular ProDH from strain MANF2 was obtained.

23

FT-IR spectroscopy is a powerful tool to provide information about the conformational and structural dynamics of a macromolecule [29]. FT-IR spectrum of the purified protein indicates strong peaks, suggesting the presence of varied functional groups, corresponding to protein and peptide bonds. The presence of different peaks in the protein was supposed to be obtained through stretching and bending vibrations in the region of infrared radiation. Proteins synthesized in bacteria under mild stress of antimicrobial agents such as A. sativum and streptomycin sulfate had shown pronounced activities against human pathogens

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[14]. In this context, we have taken further significant attempt to synthesize protein in coagulase-negative Staphylococcus sp. under mild stress of M. piperita L. for its in vitro antitubercular and anticancer properties. The purified protein exhibited promising anti-tubercular

-p

activity in a dose dependent manner. Different pH treatments affected significantly (P<0.05) the anti-tubercular activities of purified protein. Variations in the anti-tubercular properties at

re

lower or higher pH may be due to the denaturation of the purified protein as electrostatic

lP

repulsion of intermolecular and breaking of hydrogen bonds [30]. Further, the protein lost its anti-tubercular activity at high temperature (>60⁰C). It clearly suggests that heat resistance

na

nature of protein depends on its stable structural characteristics. The anti-tubercular traits of purified protein were lost completely after treatment with pepsin, trypsin, and protease K but its anti-tubercular attribute was retained by α-amylase treatment. Findings clearly represent

ur

that the purified product is proteinaceous in nature constituting amino acid residues, without

Jo

carbohydrate moieties. Similar observations were reported by Yi et al. [30] too. Most of the conventional therapies lack cancer selectivity, which can have severe

unintended complications on healthy body tissue. In spite of being a contentious therapy, the last two decades have seen a significant renaissance of bacterium-mediated cancer treatment [7]. Although promising, the present-day therapeutic bacterial agents have not revealed satisfactory outcomes and effectiveness. Hence, we had undertaken further attempt to

24

investigate new class of fermented food associated bacteria for its in vitro anticancer characteristics. In the present investigation, in vitro cytotoxicity of purified protein of strain MANF2 was determined against lung and colon cancer cells using MTT assay. The protein revealed comparatively higher anticancer activity against A549 cancer cells as compared to HT-29 cancer cells in a concentration dependent manner. Findings of the present study indicated the first report on the anticancer attribute of S. hominis associated protein synthesized under mild stress of M. piperita L. Thus, the protein synthesized in bacteria

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under mild stress conditions of certain antimicrobials may be used as new protein-based antitubercular and anticancer agents in future. 5. Conclusions

-p

In this study, growth medium containing yeast extract (0.5% w/v) and glycerol (1.5%

re

v/v), being supplemented with M. piperita L. (1.5% v/v) at log phase of strain MANF2 revealed maximum protein yield of 9.4±0.4 mg/mL using CCD of RSM. Thus, the protein

lP

production from bacteria can be enhanced by supplementing the particular doses of M. piperita L. at specific growth phase of bacteria. Further, the study represented the first report

na

on the identification of anti-tubercular and anticancer protein of 51293 Da from S. hominis under mild stress of M. piperita L. The purified protein showed sequence homology with

ur

ProDH, thereby categorizing it as new class of ProDH-like protein in S. hominis. In a nutshell, protein purified from strain MANF2 under mild stress of M. piperita L. can be used

Jo

as an ideal therapeutic agent against TB and cancer (lung and colon).

AUTHOR STATEMENT We wish to confirm that there are no known conflicts of interest associated with this publication. We confirm that the manuscript has been revised and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the revised manuscript has been 25

approved by all of us. With the submission of this revised manuscript we would like to undertake that the above mentioned manuscript has not been published elsewhere, accepted for publication elsewhere or under editorial review for publication elsewhere.

Conflict of interest None declared

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Acknowledgement Authors acknowledge Maulana Azad National Fellowship (F1-17.1/2015-16/MANF-

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2015-17-BIH-60730), University Grants Commission, Delhi, India for the support.

1.

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environment, Pol. J. Environ. Stud. 22 (2013) 1577-1587. A. Khusro, D. Sankari, Synthesis and estimation of total protein in Bacillus subtilis

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biological properties, Microb. Pathog. 132 (2019) 222-229. L. Yi, Y. Dang, J. Wu, L. Zhang, X. Liu, B. Liu, et al., Purification and

koumiss

from

Xinjiang,

China,

J.

Jo

from

ur

characterization of a novel bacteriocin produced by Lactobacillus crustorum MN047 isolated

29

Dairy

Sci.

99

(2016)

7002–7015.

ur

na

lP

re

-p

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Fig. 1: MIC determination of M. piperita L. against strain MANF2. Control represents the growth of bacteria in the absence of M. piperita L.

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Fig. 2: Growth curve (lag and log phase) analysis of strain MANF2. The lag and log phase of the bacterium was achieved at 2 and 9 h.

30

ro of

Jo

ur

na

lP

re

-p

Fig. 3: Molecular mass determination of purified protein using SDS-PAGE. Lane 1 – ammonium sulphate precipitated sample, Lane 2 – dialysed sample, Lane 3 – purified sample showing a single homogeneous band corresponding to the molecular mass of approximately 52 kDa, indicated by yellow arrow, and M – molecular mass marker

31

ro of -p re lP

Jo

ur

na

Fig. 4: MALDI-TOF MS/MS spectrum of purified protein. Each peak in the spectrum represents peptides present in the tryptic digest of purified protein. The size of the peptide ion varies generally from m/z 1689.63 to 3555.06.

32

ro of

na

lP

re

-p

Fig. 5: FT-IR spectrum of purified protein.

Jo

ur

Fig. 6: Anti-tubercular activities of purified protein and rifampicin at various concentrations (10–50 µg/mL). Values are represented as mean±SD of experiments carried out in triplicate (n = 3). abcdeValues with different letters are significantly (P<0.05) different.

33

ro of -p re lP

Jo

ur

na

Fig. 7: Morphological variations and reductions in the viabilities of A549 cancer cells in the presence of various concentrations (10–50 µg/mL) of purified protein. Yellow arrow indicates morphological changes in the cancer cells. Values are represented as mean±SD of experiments carried out in triplicate (n = 3). abcdeValues with different letters are significantly (P<0.05) different.

34

ro of -p re lP

Jo

ur

na

Fig. 8: Morphological variations and reductions in the viabilities of HT-29 cancer cells in the presence of various concentrations (10–50 µg/mL) of purified protein. Yellow arrow indicates morphological changes in the cancer cells. Values are represented as mean±SD of experiments carried out in triplicate (n = 3). abcdeValues with different letters are significantly (P<0.05) different.

35

ro of

Jo

ur

na

lP

re

-p

Fig. 9: Pathway of proline metabolism. Proline is synthesized from glutamate starting with the enzymes glutamate kinase (GK) and γ-glutamyl phosphate reductase (GPR), which in plants and animals are fused together in the bifunctional enzyme ∆1-pyrroline-5-carboxylate synthetase (P5CS). The intermediate, γ-glutamate-semialdehyde (GSA) spontaneously cyclises to ∆1-pyrroline-5-carboxylate (P5C), which is then reduced to proline by P5C reductase (P5CR). Alternatively, proline can be formed from ornithine, which is converted into P5C/GSA via ornithine-δ-aminotransferase (OAT). P5C is then reduced to proline by P5C reductase (P5CR).

36

ro of

Jo

ur

na

lP

re

-p

Fig. 10: Metabolism of proline to glutamate through the catalytic reaction of ProDH. The first enzymatic step transforms proline to ∆1-pyrroline-5-carboxylate (P5C), which is nonenzymatically hydrolyzed to glutamic semialdehyde. The semialdehyde is oxidized in the second enzymatic step to glutamate by the catalytic action of ∆1-pyrroline-5-carboxylate dehydrogenase.

37

ro of -p re

Jo

ur

na

lP

Fig. 11: Proposed hypothetical pathway for ProDH production from strain MANF2 under mild stress of M. piperita L. Proline was synthesized in M. piperita L. from glutamate through proline metabolism pathway. M. piperita L. at mild concentration exposed strain MANF2 to proline which caused the secretion of bioactive proteins from bacterium into the medium for its degradation. Proline was degraded by ProDH which was purified further through step-wise purification techniques.

38

Table 1: MIC and sub-MIC values of M. piperita L. against strain MANF2.

Strain MANF2

100.0

-

50.0

-

33.33

-

25.0

-*

20.0

+

16.67

+

ro of

M. piperita L. concentration (%)

14.29

+

12.5

+**

11.11

+ +

-p

10.0 9.09

+

8.3

+

re

5.0 4.7

lP

2.5 1.0

+ + + +

ur

na

[Note: - = No growth, + = Growth, -* = MIC, +** = Sub-MIC]

Jo

Table 2: Variables with their ranges and levels for CCD. Code

Yeast extract

A

% w/v

0.02

0.05

0.1

0.5

1.0

Glycerol

B

% v/v

0.5

1.0

1.5

2.0

2.5

M. piperita L.

C

% v/v

0.2

0.5

1.0

1.5

2.0

Variables

Unit

Range and levels

39

-α

-1

0

+1

+α

Table 3A: Experimental and predicted values of protein production from strain MANF2 under mild stress of M. piperita L. provided at lag phase. Protein content (mg/mL) Run order

Yeast extract

Glycerol (B)

Experimental

Predicted

(C)

0

-α

0

3.8

3.6

2

0

0

0

3.4

3.04

3

0

0

0

3.4

3.04

4

0

0

-α

2.6

2.2

5

+1

0

0

5.4

5.2

6

0

0

0

2.9

3.04

7

0

0

0

2.8

3.04

8

-α

0

0

2.2

1.9

9

0

+α

0

1.9

1.9

10

0

0

0

2.8

3.04

11

-1

-1

+1

2.8

2.7

12

+1

-1

-1

4.2

4.2

13

-1

+1

+1

3.4

3.3

14

+1

0

+1

6.4

6.2

15

-1

-1

-1

1.7

2.01

16

+1

+1

-1

1.6

1.6

17

0

0

0

2.9

3.04

18

0

0

+α

5.7

5.9

19

+α

0

0

5.3

5.4

20

-1

+1

-1

1.8

1.9

Jo

ur

na

lP

re

ro of

1

-p

(A)

M. piperita L.

Table 3B: ANOVA for protein production from strain MANF2 under mild stress of M. piperita L. provided at lag phase. Source

Sum of Squares df Mean Square F-value

Model

36.42

9

4.05

A-A

13.61

1

13.61

B-B

2.90

1

2.90 40

P-value

48.47 <0.0001 significant 163.01 <0.0001 34.77

0.0002

C-C

15.21

1

15.21

AB

2.43

1

2.43

29.14

0.0003

AC

2.0

1

2.0

23.90

0.0006

BC

0.1752

1

0.1752

2.10

0.1780

A²

0.7682

1

0.7682

9.20

0.0126

B²

0.1227

1

0.1227

1.47

0.2533

C²

1.99

1

1.99

23.81

0.0006

Residual

0.8348 10

0.0835

Lack of Fit

0.4214

5

0.0843

Pure Error

0.4133

5

0.0827

1.02

0.4917 not significant

ro of

Cor Total

182.20 <0.0001

37.25 19

R2 - 0.9776, Adj R2 - 0.9574, Predicted R2 - 0.8834, CV – 8.62%, Adeq precision – 22.52, df - degree of

Jo

ur

na

lP

re

-p

freedom, Significant – P < 0.05, Non-significant – P > 0.05.

41

Table 4A: Experimental and predicted values of protein production from strain MANF2 under mild stress of M. piperita L. provided at log phase. Protein content (mg/mL) Run order

Yeast extract

Glycerol (B)

Experimental

Predicted

(C)

0

-α

0

6.8

6.6

2

0

0

0

6.4

6.1

3

0

0

0

6.4

6.1

4

0

0

-α

4.6

4.5

5

+1

0

0

8.4

8.1

6

0

0

0

5.9

6.1

7

0

0

0

5.8

6.1

8

-α

0

0

4.2

4.3

9

0

+α

0

4.3

4.6

10

0

0

0

5.8

6.1

11

-1

-1

+1

5.8

5.6

12

+1

-1

-1

7.2

7.2

13

-1

+1

+1

6.4

6.2

14

+1

0

+1

9.4

9.3

15

-1

-1

-1

4.1

4.2

16

+1

+1

-1

4.3

4.2

17

0

0

0

5.9

6.1

18

0

0

+α

8.7

8.9

19

+α

0

0

8.3

8.4

20

-1

+1

-1

4.2

4.1

Jo

ur

na

lP

re

ro of

1

-p

(A)

M. piperita L.

Table 4B: ANOVA for protein production from strain MANF2 under mild stress of M. piperita L. provided at log phase. Source Model

Sum of Squares df Mean Square F-value

P-value

49.9

9

5.47

A-A

19.41

1

19.41

236.92 <0.0001

B-B

4.36

1

4.36

53.22 <0.0001

42

66.73 <0.0001 significant

C-C

22.80

1

22.80

AB

2.97

1

2.97

36.31

0.0001

AC

1.33

1

1.33

16.22

0.0024

BC

0.1928

1

0.1928

2.35

0.1559

A²

0.1955

1

0.1955

2.39

0.1534

B²

0.2432

1

0.2432

2.97

0.1156

C²

0.9517

1

0.9517

11.62

0.0067

Residual

0.8191 10

0.0819

Lack of Fit

0.4057

5

0.0811

Pure Error

0.4133

5

0.0827

0.9816

0.5079 not significant

ro of

Cor Total

278.31 <0.0001

50.01 19

R2 - 0.9836, Adj R2 - 0.9689, Predicted R2 - 0.9208, CV – 4.66%, Adeq precision – 26.29, df - degree of

-p

freedom, Significant – P < 0.05, Non-significant – P > 0.05.

Purification step

Volume

re

Table 5: Purification steps of protein from strain MANF2. RLU (%) Total

Purification

98.8

12535.3

0.0079

1

100

84.3

9546.3

0.009

1.14

85.3

(mL)

protein

Specific activity

fold

Yield (%)

1000

Ammonium

1000

sulphate

Dialysis

10

78.1

340.3

0.23

29.1

79.1

2

71.5

6.6

10.84

1372.1

72.3

ur

precipitation

na

CFNS

Jo

DEAE –Cellulose

lP

(mg)

column

chromatography

Table 6: Effect of pH, temperature, and enzymes on residual anti-tubercular activities of protein. Treatment

Residual activity (%)

43

Control

100±0.5a

3

40.4±0.4f

4

44.8±0.4e

5

54.6±0.5d

6

93.5±0.5c

7

96.6±0.4b

8

54.5±0.4d

Temperature 100±0.5a

40⁰C, 2 h

97.2±0.4b

60⁰C, 1 h

54.5±0.4c

80⁰C, 30 min

26.8±0.5d

Enzymes

na

Control

14.4±0.5e 10.8±0.4f

lP

121⁰C, 15 min

re

100⁰C, 20 min

-p

Control

100±0.5a

Pepsin

0.00±0.00

Trypsin

0.00±0.00

ur

ro of

pH

0.00±0.00

α-amylase

97.8±0.5b

Jo

Protease K

Values represent mean±SD of experiments carried out in triplicate (n = 3). Means followed by different superscripts are significantly different (P<0.05).

a,b,c,d,e,f

44